Low Serum Creatinine Levels in Early Pregnancy Are Associated with a Higher Incidence of Postpartum Abnormal Glucose Metabolism among Women with Gestational Diabetes Mellitus: A Retrospective Cohort Study

The predictive factors for the progression from gestational diabetes mellitus (GDM) to type 2 diabetes remain incompletely elucidated. Our objective was to investigate the link between serum creatinine, a proxy for skeletal muscle mass, and the development of postpartum abnormal glucose metabolism (AGM). Methods: A retrospective review of the medical records of 501 women with GDM was conducted, all of whom underwent a 75 g oral glucose tolerance test (OGTT) between 4 and 12 weeks postpartum. Women were grouped based on quartiles of serum creatinine at the first antenatal visit to estimate the association between serum creatinine and postpartum AGM incidence. Results: Compared with the highest quartile of creatinine, lower quartiles were substantially linked to an increased incidence of postpartum AGM (adjusted odds ratios 3.37 [95% CI 1.77–6.42], 2.42 [95% CI 1.29–4.51] and 2.27 [95% CI 1.23–4.18], respectively). The generalized additive model suggested a linear relationship between serum creatinine levels and the risk of postpartum AGM below 68 µmol/L of serum creatinine levels. A decrease of 2 μmol/L in serum creatinine levels was found to be associated with a 10% increase in the odds of developing postpartum AGM. Linear regression revealed that a low serum creatinine level was linked to a higher postpartum 2-h glucose level and a decreased insulinogenic index (p = 0.007 and p = 0.027, respectively). Conclusions: An association was observed between lower serum creatinine levels in early pregnancy and an increased risk of postpartum AGM and poorer β-cell function in women with a recent history of GDM. Further research is needed to understand the mechanisms underlying our findings, as well as the role of skeletal muscle mass or nutritional status in early pregnancy on later glucose metabolism.


Introduction
Gestational diabetes mellitus (GDM), defined as glucose intolerance first identified during the second and third trimesters of pregnancy, affects 3-14% of pregnancies worldwide [1]. Multiple studies have revealed that insulin sensitivity is lowered by roughly 50-60% in the third trimester compared to pre-pregnancy [2][3][4]. GDM develops when a woman's pancreas fails to secrete enough insulin in response to significantly decreased insulin sensitivity, resulting in varying degrees of hyperglycemia [5]. Women with a prior incidence of GDM are facing a much greater possibility of encountering type 2 diabetes mellitus (T2DM) postpartum, with a 7-to 10-fold increase compared to those who had chronic hypertension; (4) viral infection or positive carrier status; (5) cancer; (6) connective tissue diseases; (7) uncontrolled endocrine disorders; or (8) multiple gestation. Women with incomplete data and no follow-up examination were also removed. Ultimately, the analysis's final sample size was 501 women.

Data Collection
To obtain patient data, the medical record system of the hospital was examined. Personal information, age, height, pre-pregnancy weight, pre-pregnancy BMI, gestational weight gain, insulin use, parity, familial T2DM history, medication use, previous GDM history, and other disease history were all gathered. The baseline laboratory test results collected included fasting plasma glucose (FPG), serum creatinine, aspartate transaminase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and γ-glutamyl transpeptidase (GGT) during the first antenatal visit at 10-14 weeks of gestation. Lipid data from middle pregnancy (24-28 weeks) were also included. Standardized methods were used to analyze blood samples collected in the morning after a minimum 8 h overnight fast.

Definitions
At 24-28 weeks of gestation, all women had a 75 g OGTT performed as a standard check for GDM (1). In line with the standards set forth by the International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria [31], the GDM diagnosis was established if any of the three criteria below were fulfilled following a 75 g OGTT: FPG levels ≥ 92 mg/dL, 1-h glucose levels ≥ 180 mg/dL, and 2-h glucose levels ≥ 153 mg/dL.
As an attempt to define dyslipidemia in the second trimester of pregnancy, the recommended values for the maternal serum lipid profile in China were consulted: total cholesterol (TC) ≥ 7.50 mmol/L, triglycerides (TGs) ≥ 3.56 mmol/L, high-density lipoprotein cholesterol (HDL-C) ≤ 1.41 mmol/L, and low-density lipoprotein cholesterol (LDL-C) ≥ 4.83 mmol/L [34].

Statistical Analysis
The estimation of required minimum sample size was using software G*Power version 3.1 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, German). The testing method was F test, the effect size f was set at a medium level of 0.25, type I error (alpha) 0.05, power 0.80, and the independent group number was 4. After calculation, the minimum required sample size of a single group was 45. Considering possible dropout and small effect size, we doubled the sample size of each group to 90. Therefore, at least 360 participants would be included.
By using one-way ANOVA, normally distributed continuous variables were compared and given as the mean and standard deviation. The Mann-Whitney U test was used to compare non-normally distributed data, which were presented as the median with interquartile range (IQR). The frequencies and percentages of categorical variables were calculated and compared using the Chi-square test. The study population was divided into quartiles according to their baseline serum creatinine levels, and the difference in postpartum AGM incidence between quartiles was evaluated using Che chi-square test. To determine the connection between serum creatinine and the incidence of postpartum AGM, odds ratios (ORs) and 95% confidence intervals (Cis) were generated using logistic regression models. Linear regression worked to identify relations of log-transformed creatinine in serum with log-transformed postpartum fasting glucose, 2-h glucose, insulinogenic index, and HOMA-IR. p < 0.05 was deemed to be statistical significance. SPSS version 22.0 (IBM Corp, Armonk, NY, USA) was used for statistical analyses. In addition, the general additive model served to detect the shape of the connection between continuous creatinine and the risk of postpartum AGM; these analyses were carried out with the use of the statistical packages R (The R Foundation; http://www.r-project.org (accessed on 10 September 2020); version 3.6.3) and EmpowerStats (www.empowerstats.net (accessed on 2 July 2022), X&Y solutions, Inc. Boston, MA, USA).

Baseline Characteristics
The study cohort comprised 501 women with a recent diagnosis of GDM, with a median age of 33 years (IQR, 30-37 years), a median BMI of 21.5 kg/m 2 (IQR, 19.8-23.4 kg/m 2 ), a median serum creatinine level of 42 µmol/L (IQR, 46-50 µmol/L), and a median FPG of 77.4 mg/dL (IQR, 72-82.8 mg/dL) at 10-14 weeks gestation. Table 1 presents baseline parameters of the research population's demographic, clinical, and metabolic traits during pregnancy per first-trimester serum creatinine quartile. By comparison to women with higher serum creatinine levels, those with lower levels tended to be younger, with lower height and weight before pregnancy and lower BUN in early pregnancy. Other risk factors, such as FPG, ALT, AST, and GGT at baseline and lipid profile at 24-28 weeks, did not differ significantly among the serum creatinine quartiles. Compared with the women in the highest creatinine quartile, those with lower creatinine levels appeared to have higher fasting and 2-h OGTT glucose levels during 24-28 weeks of pregnancy, although this difference was not significant.

Association between Serum Creatinine Quartiles in Early Pregnancy and Postpartum AGM
Among the 501 women with GDM included in the analysis, 170 (33.9%) women developed postpartum AGM, of whom 3.8% had DM, 30.1% had IGT, and no women had isolated IFG. Figure 1 indicates the incidence of postpartum AGM (including IGT and DM) in different serum creatinine quartiles. The lowest incidence of postpartum AGM was observed in the highest quartile of serum creatinine, with only 20.9% of women developing the condition, compared to 41.5%, 37.9%, and 34.2% in the lowest to third quartile, respectively (Chi-square test, p = 0.007).

Association between Serum Creatinine Quartiles in Early Pregnancy and Postpartum AGM
Among the 501 women with GDM included in the analysis, 170 (33.9%) women developed postpartum AGM, of whom 3.8% had DM, 30.1% had IGT, and no women had isolated IFG. Figure 1 indicates the incidence of postpartum AGM (including IGT and DM) in different serum creatinine quartiles. The lowest incidence of postpartum AGM was observed in the highest quartile of serum creatinine, with only 20.9% of women developing the condition, compared to 41.5%, 37.9%, and 34.2% in the lowest to third quartile, respectively (Chi-square test, p = 0.007).

Association of Serum Creatinine and Postpartum AGM Incidence: Subgroup Analyses
For further analysis, we stratified the women by maternal age (median age of 33 years), BMI (overweight is BMI ≥ 23 kg/m 2 ) (according to Asia-specific standards from the WHO), and dyslipidemia. Stratified analysis revealed consistent significant associations between serum creatinine and postpartum AGM across all subgroups. The fully adjusted model demonstrated that women in the lowest creatinine quartile are exposed to an increased risk of postpartum AGM by comparison to those in the highest creatinine group in all subgroups. The middle two quartiles were linked to a higher potential of postpartum AGM, only in the higher age group, the lower BMI group, and the dyslipidemia group (Table 3).

Association of Continuous Serum Creatinine Levels and Postpartum AGM Incidence
A generalized additive model (GAM) was utilized to build a smooth curve and detect the relationship between serum creatinine and the risk of postpartum AGM. In light of the unadjusted GAM analysis, a linear correlation was observed between serum creatinine levels and postpartum AGM risk below a level of 68 µmol/L (upper limit in the women in our study) and the risk of postpartum AGM (Figure 2).

Association of Continuous Serum Creatinine Levels and Postpartum AGM Incidence
A generalized additive model (GAM) was utilized to build a smooth curve and detect the relationship between serum creatinine and the risk of postpartum AGM. In light of the unadjusted GAM analysis, a linear correlation was observed between serum creatinine levels and postpartum AGM risk below a level of 68 µmol/L (upper limit in the women in our study) and the risk of postpartum AGM (Figure 2).  Considering serum creatinine level as a continuous variate (Table 4), the logistic regression model revealed that each 2 µmol/L decrease in serum creatinine was linked to 8% higher odds of postpartum AGM below a serum creatinine threshold of 68 µmol/L at 10-14 weeks gestation in women with GDM. Similarly, in terms of the age-adjusted model as well as the fully adjusted model, the logistic regression model revealed that higher levels were linked to a decreased incidence of postpartum AGM (p = 0.001, p = 0.003, respectively). Findings from the adjusted model indicated that a decline of 2 µmol/L in serum creatinine was significantly associated with a 10% rise in the likelihood of postpartum AGM.

Continuous Serum Creatinine Levels and Postpartum Glucose Level, β-Cell Function and HOMA-IR
Calculation of β-cell function was performed, applying data from the OGTT: IGI (∆insulin (30-0 min)/∆glucose (30-0 min)). Insulin resistance was quantified by means of HOMA-IR. Linear regression was employed to investigate the correlations among logtransformed serum creatinine and log-transformed postpartum fasting glucose, 2-h glucose, IGI, and HOMA-IR ( Table 5). The investigation of linear regression indicated that the serum creatinine level was negatively associated with postpartum 2-h glucose level and positively linked to IGI. In the multivariate model, a high serum creatinine level was associated with a lower postpartum 2-h glucose level (β = −0.23, 95% CI [−0.40, −0.07], p = 0.007) and a better IGI (β = 0.55, 95% CI [−0.06, 1.04], p = 0.027). In adjusted models, the serum creatinine level did not correlate with FPG or HOMA-IR.

Discussion
In the current retrospective cohort analysis of women who have recently experienced GDM, we found that low serum creatinine levels at 10-14 weeks gestation were positively associated with an increased risk of postpartum AGM incidence, regardless of other major risk factors such as age, BMI, gestational weight increase, family history of T2DM, previous history of GDM, insulin use and glucose level. Additionally, analysis using the unadjusted GAM indicated that serum creatinine and the risk of postpartum AGM are correlated linearly. In the adjusted model, postpartum AGM risk decreased by 10% for every 2 µmol/L increase in serum creatinine in patients with GDM who had serum creatinine levels below 68 µmol/L. Serum creatine, generally considered an easily measured surrogate marker of skeletal muscle mass, has reportedly been linked to the occurrence of T2DM. N. Harita et al. first reported the link between T2DM occurrence and serum creatinine in their 4-year cohort investigation of 8570 Japanese male, revealing that lower levels of serum creatinine independently predicted progression to T2DM in nonobese Japanese men with normal creatinine levels at baseline [27]. Since then, a further cohort research study in an eastern Asian community shed light on a link between low blood creatinine levels and a higher prevalence of diabetes in both men and women, with a reverse J-shaped correlation in males and a linear relationship in women [29]. Interestingly, a cross-sectional study in Caucasian women also showed a piecemeal linear correlation of T2DM and serum creatinine, indicating that the risk of T2DM was 6% lower in women with a serum creatinine level below 69 µmol/L for every 1 mol/L increase in serum creatinine, which resembles the findings of our study [35].
Our findings extended this association specifically to pregnant women initially, providing evidence for a correlation among serum creatinine and persistent glucose metabolism disorders in women with GDM. The serum creatine level at the first antenatal visit, measured in a simple and rapid blood test, might be a potential biomarker for postpartum AGM among women with GDM. As far as we are aware, one study examined metabolites in the first trimester and found that, by comparison to women with normal glucose tolerance (NGT), those who subsequently developed GDM had higher levels of anthranilic acid, glutamate, alanine, and allantoin, along with significantly lower levels of creatinine [36]. This indirectly underpins our finding that lower levels of creatinine in early pregnancy may be linked to a higher risk of metabolic abnormalities. It would be worthwhile to further explore whether low serum creatinine levels are associated with an increased incidence of GDM.
The underlying mechanism by which lower serum creatinine levels increase the risk of postpartum AGM remains unknown in this study. However, it is possible that lower serum creatinine levels indicate decreased skeletal muscle mass, as hypothesized. In our study, women with GDM had a median pre-pregnancy BMI of just 21.5 kg/m 2 , and we discovered that early postpartum glucose metabolism disorder in these women primarily manifested as IGT rather than IFG, which is consistent with previous Asian studies [17,18,37]. Unlike individuals with IFG who are generally obese, accompanied by hepatorenal insulin resistance and elevated gluconeogenesis, individuals with IGT have reduced peripheral insulin sensitivity (mainly representing skeletal muscle), combined with impairments in glucosestimulated insulin secretion, resulting in postprandial hyperglycemia [38][39][40]. Skeletal muscle serves as a key player as the major site of postprandial glucose disposal, accounting for about 85% of glucose uptake in the postprandial state [19]. Accordingly, the increased risk of glucose intolerance associated with low serum creatinine levels may be explained by low muscle mass.
Our current study demonstrated a negative association between low serum creatinine and postpartum β-cell function, but we did not observe any association between serum creatinine and HOMA-IR, which is a measure of hepatic insulin resistance. It is important to note that individuals with impaired glucose tolerance typically exhibit muscle insulin resistance with only mild hepatic insulin resistance. Future studies should explore muscle insulin sensitivity using the hyperinsulinemic-euglycemic clamp technique. Among young women, the desire to shed weight is very common; however, inadequate muscle mass might be a potential cause for the inability to maintain normal glucose tolerance and may lead to persistent glucose abnormalities in GDM women, suggesting that aerobic and resistance exercise before and after pregnancy may contribute to the prevention of postpartum diabetes by increasing muscle mass and improving β-cell function, which should be further confirmed by prospective and multicenter studies.
Numerous variables, most notably deteriorating renal function, might affect serum creatinine levels. In this study, we excluded women with underlying kidney injury or other conditions that could affect serum creatine levels, such as prediabetes, hypertension, and connective tissue diseases, to minimize the effect of other factors on creatinine. The concentration of serum creatinine is influenced by age, gender, and body composition because creatinine is produced from muscle tissue. In this study, we adjusted for these factors and still observed a significant correlation between serum creatine and postpartum glucose disorders. In addition to the abovementioned factors, serum creatinine is also affected by nutritional status and protein intake [41]. There is evidence to suggest that dietary factors, particularly protein and the amino acid leucine, play a critical role in promoting muscle protein synthesis and preserving muscle mass [42,43]. Vegetarians have considerably lower serum creatinine concentrations than omnivores in numerous big cross-sectional investigations [44,45]. Interestingly, an animal study revealed that insufficient nutrition during the first half of pregnancy is associated with later metabolic abnormalities and favors a diabetogenic condition in the pregnant mother [46]. Further investigation is required to determine if early nutrition is related to the increased risk of glucose abnormalities associated with low serum creatinine levels. Additionally, it is important to note that glomerular hyperfiltration, which is linked to lower serum creatinine levels, has been shown to raise the risk of metabolic disorders, including diabetes [47,48]. Potential mechanisms include insulin resistance, as insulin has been shown to exert a direct effect on glomerular podocytes [47], increased renal gluconeogenesis [49], and endothelial dysfunction/chronic inflammation [50]. Serum creatinine levels also typically decrease during pregnancy due to physiologic glomerular hyperfiltration [51], while higher glomerular filtration rates have been found to increase the risk of adverse maternal/fetal outcomes [52][53][54]. Our study showed a potential association between low serum creatine in early pregnancy and persistent glucose metabolism disorders after adjusting for age, and higher glomerular filtration rates may also be a factor. However, the correlation between glomerular filtration rate and creatinine level makes it difficult to determine which is the culprit.
The reliability and validity of our findings are strengthened by the following: the serum creatinine levels were assessed during the initial antenatal visit, and prior to detecting any glucose abnormalities, women with underlying kidney disease or other conditions that might affect serum creatinine levels were excluded, the models were adjusted for multiple confounders, and standardized criteria were used in our universal screening and diagnosis. Therefore, in spite of our study's retrospective nature, this research is still of great value in demonstrating the correlation between low serum creatinine concentration and the progression of postpartum AGM. Nonetheless, the present study is not exempt from certain limitations. First, it would be preferable to have pre-pregnancy creatinine data; however, because pregnancies can sometimes be unintended, these data are challenging to collect. Prospective studies among fertile women can thus be conducted in the future. In addition, the actual measured skeletal muscle mass was not analyzed in this study; thus, more far-reaching research is demanded to shed light on mechanisms underlying our findings.

Conclusions
The findings of this investigation shed new light on the link between low serum creatinine levels in early pregnancy and the increased incidence of postpartum AGM, suggesting that low serum creatine levels at the first antenatal visit, measured in a simple and rapid blood test, might be a potential biomarker for postpartum AGM among women with GDM. The inverse correlation between serum creatinine and persistent glucose metabolism disorders might reflect the protective effect of skeletal muscle on glucose metabolism. Further research is required to understand the mechanisms underlying our findings, as well as the role of skeletal muscle mass or nutritional status in early pregnancy on later glucose metabolism.
Funding: This study was supported by grants from the 5010 Project Foundation of Sun Yat-sen University (No. 2017001).

Institutional Review Board Statement:
This study was conducted in accordance with the Declaration of Helsinki, and approved by the ethics committees for clinical research and animal trials of the First Affiliated Hospital, Sun Yat-sen University (ethical approval number: [2020] 048; Date: 14 January 2020), and the waiver of patient consent was approved because this was a retrospective study.
Informed Consent Statement: Patient consent was waived due to a retrospective, non-interventional study design; the Ethics Committee waived the need for informed consent in terms of data collection and the analysis and publication of results. After collecting clinical data, patient identifiers are deleted, and patients cannot be identified directly or indirectly later.

Data Availability Statement:
The data can be retrieved from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.